We propose to detect and characterize intermediates in the reaction cycles of a varied and versatile set of non-heme Fe- containing dioxygenases as a means to study the mechanisms of biological dioxygen activation. The most important oxygen activation strategies used in nature are represented within this group of enzymes, allowing the problem to be approached on a broad front. The activation of O2 by Fe-containing enzymes is highly regulated because escape of reactive oxygen species from the active site causes damage of genetic material and other biomolecules, leading to cell death in bacteria and diseases in humans. Consequently, our study will delineate the stabilizing and destabilizing forces for the reactive intermediates in the O2 activation process. The bacterial classes of enzymes we will study all use aromatic substrates and represent the major means by which natural and man-made aromatics are biodegraded in the environment. This provides the first line of defense against the toxic and carcinogenic effects of many of these compounds, and thus these enzymes have another substantial impact on human health. The dioxygenase enzymes proposed for study include: Fe(II)-containing extradiol dioxygenases, Fe(III)-containing intradiol dioxygenases, and redox cycling Rieske dioxygenases. We have developed a set of hypotheses for the mechanisms by which the active site iron in each type of dioxygenase is used to promote catalysis, which will now be tested by exploiting methods we have perfected to observe their reactions step by step as they occur. Past studies have led to the development of single turnover systems for each of the enzyme classes. Intermediates will be detected and trapped by coordinated use of stopped-flow transient kinetics, rapid mixing freeze quench (RFQ), site directed mutagenesis, and use of slow substrate analogs. Also, novel in crystallo techniques will be used in which the reactions are slowed (and often stopped at intermediates) by carrying them out in single enzyme crystals. The intermediates trapped by RFQ will be characterized by a range of spectroscopies including optical, EPR, rRaman, NRVS, EXAFS, NIR CD, VTVH MCD, and Mssbauer. The 3D structures of intermediate trapped in crystallo will be solved by X-ray crystallography and through the use of newly developed single crystal optical and (r)Raman spectroscopies. In preliminary studies, we have detected and trapped several widely postulated, but unobserved, intermediates in each of the enzyme classes proposed for study. These will be now be characterized as methods to detect and trap additional intermediates are developed. In parallel studies, methods to replace the Fe(II) in extradiol dioxygenases with Co(II) and Mn(II) without loss of activity have been developed. The new spectroscopic and kinetic properties of these metal-substituted enzymes promise new insight into the dioxygenase mechanism as well as the rationale for metal choice in enzymes. This work will yield fundamental information about the chemistry of oxygenases, oxygen, and metals in biological systems. The basic concepts that emerge will be useful in such areas as the mechanisms of similar enzymes in mammals and methods to interdict the production of deleterious diffusible reactive oxygen species in humans.